TRK tyrosine kinase receptor is the physiological receptor for nerve growth factor

ABSTRACT

The present invention relates to a complex comprising nerve growth factor (NGF) and trk-proto-oncogene protein. The present invention also relates to methods for detecting the presence of NGF and trk-proto-oncogene receptor. The present invention further relates to methods that can be used in diagnostics and therapeutics for neurodegenerative diseases such as Alzheimer&#39;s and Huntington&#39;s by detecting NGF-trk receptor pairs and the phosphorylation of trk protein.

This is a divisional of application Ser. No. 07/885,731, filed May 19,1992, now abandoned, which in turn is a continuation in part ofapplication Ser. No. 07/668,298, file Mar. 14, 1991, now U.S. Pat. No.5,231,001.

FIELD OF THE INVENTION

The present invention relates to a complex comprising the neurotrophicfactors: nerve growth factor (NGF) and trk-proto-oncogene receptorprotein. The present invention also relates to methods for detecting thepresence of NGF ligand, and trk-proto-oncogene receptor protein.

The present invention further relates to methods of diagnosing andtreating conditions of nerve growth disease and regeneration such asAlzheimer's disease and neuroblastoma. In particular the present methodinvolves detection of the ligand receptor pairs.

The present invention further relates to methods for detectingneurotrophic factor receptor/ligand complexes on the basis of structuraland functional relatedness to trk and NGF.

The present invention further relates to methods for detectingphosphorylation of neurotrophic factor receptor protein.

BACKGROUND OF THE INVENTION

The development of the vertebrate nervous system is characterized by aseries of complex events beginning with an apparently homogeneousneuroepithelium in the early embryo and leading to formation of diverse,highly ordered, and interconnected neural cell types in the adult.Considerable descriptive and experimental evidence has been amassedwhich points to the existence of limiting diffusible factors that arerequired for the targeting, survival, and proper synaptic arrangement ofneurons (R. W. Oppenheim, In: Studies in Developmental Neurobiology.(Cowan, W. M. ed.), Oxford University press, pp. 74-133, 1981; W. D.Snider and E. M. Johnson, Ann. Neurol. 26:489-506 (1989)). Functionalneuronal circuits are sculpted from an initially overabundant productionof neurons during development. In the midterm embryo, a process ofprogrammed cell death eliminates a major proportion of the neuronpopulation, leaving behind the appropriate number of neurons requiredfor innervation of target tissues (V. Hamburger and R. Levi-Montalcini,J. Exp. Zool. 111:457-502 (1949); Y.-A. Barde, Neuron 2:1525-1535(1989)).

The protein growth factors of the neurotrophin family, which includesnerve growth factor (NGF), brain-derived neurotrophic factor (BDNF),neurotrophin-3 (NT-3), neurotrophin-4 (NT-4), and neurotrophin-5 (NT-5),regulate nervous system development (Y. A. Barde, Neuron 2:1525-1534(1989)); (H. Thoenen, Trends Neurosci. 14:165-170 (1991)); (J. Leibrocket al., Nature 341:149-152 (1989)); (P. Ernfors et al., Neuron 5:511-526(1990)); (A. Hohn et al., Nature 344:339-341 (1990)); (P. C.Maisonpierre et al., Science 247:1446-1451 (1990)); (A. Rosenthal etal., Neuron 4:767-773 (1990)); (K. R. Jones and L. F. Reichardt, Proc.Natl. Acad. Sci. USA 87:8060-80640 (1990)); (F. Hallbrook et al., Neuron6:845-858 (1991)) and are also believed to play an important role instructural maintenance, plasticity and repair of the adult nervoussystem (H. Thoenen et al., Rev. Physiol. Biochem. Pharmacol. 109,145-178 (1987); S. R. Whittemore and A. Seiger, Brain Res. Rev.12:439-464 (1987)); (F. Hefti et al., Neurobiol. Aging 10:515-533(1989)). At least two types of proteins are involved in the formation offunctional receptors for neurotrophin growth factors. These are the lowaffinity NGF receptor protein (p75-NGFR) (M. V. Chao et al., Science232:518-521 (1986); M. J. Radeke et al., Nature 325:593-597 (1987)) andproducts of trk related proto-oncogenes (B. L. Hempstead et al., Nature344:339-341 (1990)); (D. R. Kaplan et al., Cell 61:125-133 (1990)); (D.R. Kaplan et al., Science 252:554-558 (1991a)); (D. R. Kaplan et al.,Nature 350:158-160 (1991b)); (S. P. Squinto et al., Neuron 5:757-766(1990)); (S. P. Squinto et al., cell 65, 885-893 (1991)); (D. Soppet etal., Cell 65:895-903 (1991)); (C. Cordon-Cardo et al., Cell 66:173-183(1991)); F. Lamballe et al., Cell 66:967-979 (1991)).

The discovery of Nerve Growth Factor (NGF) provided the first directevidence for the existence of neurotrophic, polypeptide factors (R.Levi-Montalcini and V. Hamburger, J. Exp. Zool. 116:321-362 (1951); R.Levi-Montalcini and P. U. Angeletti, Phys. Rev. 48:534-569 (1968)). Thiswas followed by the more recent description of additional neurotrophicfactors: BDNF, CTNF, and NT-3 (Snider and Johnson, Ann. Neurol.26:489-506 (1989)); (G. Barbin et al., J. Neurochem. 43:1468-1478(1984)); (P. C. Maisonpierre et al., Science 247:1446-1451 (1990)). Thephysiological consequences elicited by NGF in vitro and in vivo havebeen at the center of research in neurobiology for several decades.Consequently, considerable information is now available about the celltypes that respond to NGF in the peripheral and central nervous systems.

NGF is known to play a role in the targeting and survival of sympatheticand neural crest-derived sensory neurons as well as in selectedpopulations of cholinergic neurons in the brain (L. A. Greene and E. M.Shooter, Annu. Rev. Neurosci. 3:353-402 (1980); H. Thoenen and Y.-A.Barde, Physiol Rev. 15 60:1284-1335 (1980); H. Gnahn et al., Dev. Brain.Res. 9:45-52 (1983)). It appears that the NGF dependent cholinergicneurons in the basal forebrain correspond to the population of cellsthat undergo attrition in Alzheimer's disease (F. Hefti, Annals ofNeurology, 13:109-110 (1983)); (Hefti and Wemer, (1986)); (Johnson andTanuchi, (1987)); (P. J. Whitehouse et al., Science 215:1237-1239(1982)). In vivo studies, in which NGF was injected in the periphery ofthe mouse embryo trunk, result in enhanced survival of sensory gangliathat are normally targeted for cell death (V. Hamburger et al., J.Neurosci 1:60-71 (1981)); (I. B. Black et al., In: Growth Factors andDevelopment, Current Topics in Developmental Biology, Vol. 24(Nilsen-Hamilton, ed.), pp. 161-192 (1990)).

Exposure of embryos to NGF antibodies results in reduced survival ofdorsal root ganglion neurons while injection of NGF antibodies intoneonate mice has the principal effect of inhibiting the survival ofsympathetic neurons (R. Levi-Montalcini and B. Booker, Proc. Natl. Acad.Sci. USA, 46:373-384 or 384-391 (1960); S. Cohen, Proc. Natl. Acad. Sic.USA, 46:302-311 (1960); E. M. Johnson et al., Science 210:916-918(1980)).

In vitro, some tumor cell lines of neural origin respond to the presenceof NGF by undergoing differentiation along neuronal pathways. PC12cells, derived from a rat pheochromocytoma, are the best characterizedof these cell lines and represent a widely accepted model forNGF-mediated response and for neuronal differentiation (L. A. Greene andA. S. Tischler, Proc. Natl. Acad. Sci. USA 73:2424-2428 (1976)).

Although much is understood about the biology of NGF outside the cell,the mechanisms by which NGF elicits neurotrophic effects within the cellhave not been fully resolved. Interaction of NGF with a cell receptor isa requisite step in the transmission of neurotrophic signals within thecell (see M. V. Chao, In: Handbook of Experimental Pharmacology, Vol.95/II Peptide Growth Factors and Their Receptors II (M. B. Sporn and A.B. Roberts, eds.), Springer-Verlag, Heidelberg, pp. 135-165 (1990)). Amajor advance in understanding NGF interactions with the cells was theidentification and cloning of a 75 kDa receptor (75kNGF-R) that bindsNGF, and is present in NGF-responsive cells. The clones of the geneencoding 75kNGF-R have been characterized from several species (M. V.Chao et al., Science 232:418-421 (1986); M. J. Radeke et al., Nature325:593-597 (1987)). Unfortunately, the structural and biologicalproperties of 75kNGF-R have provided limited clues about the nature ofthe NGF signal transduction pathway inside the cell. 75kNGF-R displaysthe binding properties of a low affinity NGF receptor (Kd≈10³¹ 9 M) whenexpressed in exogenous cell lines and analysis of the intracellulardomain has not revealed putative domains of catalytic action (Chao etal. (1990)).

The biological responsiveness to NGF, however, is widely held to dependupon interactions with a high affinity binding component implying thatother receptor or receptor subunits are probably involved in NGFresponses. The search for potential second messengers that mighttransmit NGF signals in PC12 cells has led to recent evidence indicatingthat activation of tyrosine kinases represent an early response to thepresence of NGF (Kaplan et al. (19991a)). These data implicate tyrosinekinases as candidates in the composition of a high affinity receptor.

The trk gene products but not the p75-NGFR exhibit protein kinaseactivity (D. Martin-Zanca et al., Mol. Cell. Biol. 9:24-33 (1989));(Kaplan et al. (1991a)). Individual neurotrophins bind to and stimulatetyrosine phosphorylation of different subsets of trk receptors. Trkbinds to NGF but not BDNF (Kaplan et al. (1991b)); (Soppet et al.(1991)), trkB binds BDNF but not NGF (Cordon-Cardo et al., (1991)).NT-3, in vitro, is capable of interacting with trk and trkB receptorsand with trkC (Cordon-Cardo et al., (1991)); (Lamballe et al., (1991));(Soppet et al., (1991)); (L. R. Berkemeier et al., Neuron 7:857-866(1991)).

The trk proto-oncogene encodes a tyrosine kinase (TK) receptor with atightly regulated neural pattern of expression during murine development(D. Martin-Zanca et al., Genes Dev. 4:683-694 (1990); D. Martin-Zanca etal., In: The Avian Model in Developmental Biology: From Organism toGenes, Editions du CNRS-1990, pp. 291-302 (1990)). In vivo, transcriptsfor this gene were observed only in neural crest-derived sensory neuronsof the peripheral nervous system through E17 of mouse development.Applicants have investigated the possible involvement of trk in pC12cell NGF-mediated events.

Cholinergic neurons of embryonic rat basal forebrain in culture areknown to respond to NGF, brain derived neurotrophic factor (BDNF), basicfibroblast growth factor (bFGF), insulin and the insulin-like growthfactors I and II (G. Ferrari et al., Develop. Biol. 133:140-147,(1989)); (B. Knusel et al., Exper. Neurol. 110:274-383 (1990a)); (B.Knusel et al., J. Neurosci. 10:558-570 (1990b)); (B. Knusel et al.,Proc. Natl. Acad. Sci. USA 88:961-965 (1991)); (Alderson et al.,(1990)). Recently, it has been found also that treatment with NT-3,albeit only at very high concentration of this neurotrophin, increasesthe activity of the cholinergic marker enzyme choline acetyltransferase(CHAT) in these cultures (unpublished observations). While BDNF, bFGFand the insulin family of growth factors also stimulate centraldopaminergic and, possibly GABAergic neurons (GABA being τ-amino butyricacid, an amino acid neurotransmitter), stimulation by NGF and NT-3 seemto be selective to the basal forebrain cholinergic neuron (Knusel etal., (1990a), (1991) and unpublished).

K-252a and K-252b, two related alkaloid-like compounds from microbialorigin and known to interfere with protein kinase activities incell-free systems, inhibit several biological actions of NGF (H. Kase etal., Biochem. Biophys. Res. Commun. 142:436-440 (1987)); (S. Koizumi etal., J. Neurosci. 8:715-721 (1988)); (Y. Matsuda and J. Fukuda,Neurosci. Lett. 87:295-301 (1989)). K-252a prevents the NGF inducedmorphological transformation of proliferating PC12 pheochromocytomacells into neuron-like cells and inhibits the NGF stimulated growthfactor phosphorylation of proteins, but not the basic fibroblast growthfactor (bFGF) or epidermal growth factor (EGF) stimulatedphosphorylation of selected proteins (D. S. Smith et al., J. Neurochem.53:800-806 (1989)). K-252a, in absence of exogenously provided trophicproteins, has also been shown to exhibit neurotrophic-like activity inchick dorsal root ganglion (DRG) cultures (Borasio, 1990) and ondopaminergic neurons of the ventral mesencephalon in culture (B. Knuseland F. Refti, J. Neurochem. 57:955-962 (1991)).

It was recently demonstrated that K-252a and K-252b inhibit NGF mediatedactions on cholinergic neurons in cell culture (Knusel and Hefti,(1991)). Both compounds completely and selectively prevented the trophicaction of NGF on these cells. K-252b was effective over a wide range ofconcentrations without being cytotoxic or neurotrophic for the cultures.The discovery of the protein family of neurotrophins prompted applicantsto investigate whether K-252b inhibits biological actions ofneurotrophins other than NGF. Applicants found that K-252b selectivelyinhibits growth factors of the neurotrophin family but, surprisingly, atlower concentrations, potentiates NT-3 actions. K-252b enhanced thetrophic activity of NT-3 on primary neurons and PC12 cells andstimulated the NT-3 mediated tyrosine phosphorylation of trk in PC12cells, while inhibiting similar effects of NGF. The structurally closelyrelated NGF inhibitor K-252a, but not the similarly relatedstaurosporine, also potentiated NT-3. These results suggest that K-252bstimulates and inhibits selective cellular effects of the neurotrophinsand that these actions of K-252b are due to direct or indirectinteraction with the trk signal transduction pathway.

Others have tested recombinant human NGF, BDNF, NT-3, in presence orabsence of K-252b, by adding then to primary cultures of fetal rat basalforebrain or ventral mesencephalon, containing cholinergic anddopaxinergic neurons, respectively. A broad range of concentrations ofK-252b were tested with 50 ng/ml NGF, 200 ng/ml BDNF and 1 μg/ml NT-3,growth factor concentrations producing maximal trophic actions on thebasal forebrain cholinergic neurons (Knusel et al., 1990b, 1991 andunpublished observations).

BDNF, but not NGF or NT-3, trophically acts on dopaminergic neurons asreflected by an increase in the uptake rate for dopamine by these cells(C. Hyman et al., Nature 350:230-233 (1991)); Knusel et al. (1991)).Similar to its actions on cholinergic neurons, K-252b prevented theincrease in dopamine uptake rate mediated by BDNF. Dopamine uptake isalso stimulated by other growth factors including bFGF, epidermal growthfactor, insulin, insulin-like growth factor-1 and insulin-like growthfactor-2 (Ferrari et al., (1989)); Knusel et al., (1990a)). It wasearlier shown that K-252b does not inhibit the stimulatory action ofbFGF and insulin (Knusel and Hefti (1991)).

The need exists in the field to determine whether trk proto-oncogenetyrosine kinase receptor is activated via direct interaction with NGF.The need also exists for a means of assaying compounds which modulatethe differentiation and survival of neurons.

The present invention provides a complex comprising NGF ligand andtrk-proto-oncogene receptor. The direct binding of NGF to the &*receptor leads to tyrosine phosphorylation and tyrosine kinase activityin response to NGF exposure in trk expressing cells. Knowledge of thetrk physiological receptor and cognate NGF complex will allow a detailedstudy of nerve growth and regeneration. Furthermore, the demonstrationof NGF-trk receptor complexes demonstrates methods for identifyingrelated tyrosine kinase receptors providing additionalneurotrophic-factor pairs.

Applicants have found that a target of compounds mediating neurotrophiceffects is the neurotrophic factor receptor trk. The trk family ofreceptors binds and mediates the activity of several factors inducingthe differentiation and survival of various populations of neurons,including NGF, NT3, and NT5. Applicants have found that certaincompounds, such as the alkaloid K-252b, stimulate neuronal celldifferentiation by directly affecting the activity of ligand-activatedtrk.

Presently, compounds affecting neuronal differentiation and survival areassayed directly on primary neurons cultured from rat, mouse, orchicken. These systems are labor intensive and require the use ofanimals and expensive reagents to ensure the survival of cultured cells.In addition, the effects of the compounds are observed only afterseveral days to weeks. In the present invention, the effects of thecompounds can be observed in minutes to hours. The described systems donot require animals or expensive reagents for cell culture and can beperformed in cell-free assays.

SUMMARY OF THE INVENTION

It is an object of the present invention to provide a complex comprisinga nerve growth factor (NGF) ligand and trk-proto-oncogene proteinreceptor and methods of utilizing the complex.

In one embodiment the present invention relates to a complex of NGF andtrk-proto-oncogene receptor protein wherein said complex is free ofprotein with which it is naturally associated.

In another embodiment the present invention relates to a complexcomprising a NGF ligand and trk-proto-oncogene receptor protein whereinone member of said complex is bound to a solid support.

In yet another embodiment the present invention relates to a method ofdetecting the NGF: trk-proto-oncogene receptor protein complex in aample comprising reacting said sample with an antibody that bindsspecifically with either NGF or trk-proto-oncogene receptor protein onthe complex. A positive immunological reaction is indicative of thepresence of the complex in the sample.

In a further embodiment, the present invention relates to a method ofdiagnosing degenerative neuronal diseases in a patient suspected ofhaving the disease comprising reacting a biological sample from thepatient with an antibody that binds with NGF: trk-proto-oncogenereceptor protein complex.

In yet another embodiment, the present invention relates to a method ofdiagnosing a tissue undergoing neuronal regeneration in a patientcomprising reacting a biological sample from the patient with anantibody that binds to a NGF: trk-proto-oncogene receptor proteincomplex.

A further embodiment of the present invention relates to a method ofdiagnosing a disease state in a patient suspected of having the stateddisease comprising reacting a biological sample from the patient with anantibody that binds to a NGF: trk-proto-oncogene receptor proteincomplex.

In another embodiment, the present invention relates to a method fordetecting NGF in a sample comprising contacting the sample withtrk-proto-oncogene receptor protein under conditions such that bindingof NGF present in the sample to the receptor is effected and detectingthe presence of bound NGF.

In a further embodiment the present invention relates to a method fordetecting trk-proto-oncogene receptor protein in a sample comprising thesteps of contacting the sample with NGF under conditions such thatbinding of said receptor present in the sample to NGF is effected anddetecting the presence of bound receptor.

In an additional embodiment the present invention relates to a methodfor measuring nerve regeneration in trk proteins by measuring trkphosphorylation comprising the steps of adding a compound to said trkproteins in the absence or presence of a neurotrophic factor;precipitating the trk proteins with antibodies that recognize trkproteins; subjecting the precipitated trk proteins to electrophoresis;transferring the electrophoresed trk proteins onto a membrane; andmeasuring the amount of phosphotyrosine on the membrane.

In a further embodiment, the present invention relates to a method formeasuring nerve regeneration in trk proteins by measuring trkphosphorylation comprising the steps of combining a compound with trkprotein, a neurotrophic factor and ³² P labeled ATP to form a reactionmixture; incubating the reaction mixture; removing excess ³² P labeledATP from the mixture; subjecting the mixture to gel electrophoresis; andmeasuring the amount of phosphotyrosine on the gel.

Various other objects and advantages of the present invention willbecome apparent from the drawings and the following description of theinvention.

The entire contents of all publications mentioned herein areincorporated by reference.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the tyrosine phosphorylation of p140trk in PC12 cells andtrk-expressing NIH-3T3 cells treated with NGF. Shown in a is p140 trkimmunoprecipitated from PC12 cells or trk-expressing 3T3 cells labeledwith .sup.³² P-orthophosphate. Immunoprecipitates were prepared withrabbit anti-trk antisera 43-4 (D. Martin-Zanca et al., Mol. Cell. Biol.9:24-33 (1988)) from lysates prepared from 3T3 cells expressing the rattrk gene (trk 3T3) (lane 1) or PC12 cells treated for 5 minutes with 100ng/ml NGF at 37° C. (lanes 2 and 4) or mock treated (lane 3). Theimmunoprecipitate shown in lane 2 was prepared in the presence of thepeptide used to generate the rabbit 43-4 trk antibody (Martin-Zanca etal. (1988)). Shown in b is the phosphoamino acid analysis of trkproteins phosphorylated in vitro in p140trk immunoprecipitates from NGFtreated PC12 cells in vivo from NGF treated (+) or untreated (-) PC12cells, or in vivo from trk3T3 cells. The positions of phosphoserine (S),phosphothreonine (T), and phosphotyrosine (Y) are indicated. Shown in care the trk proteins from trk 3T3 cells phosphorylated in vivo or invivo. In lanes 1-3, p140trk immunoprecipitates were probed with P-tyrantibodies. In lanes 4-6, p140trk proteins were phosphorylated in vitroin kinase assays. Cells were treated with suramin (lanes 2 and 5) orwith 500 ng/ml NGF for 10 minutes following suramin treatment. The bandmigrating at 110 kDa is a glycosylation precursor of p140^(prototrk)(Martin-Zanca et al. (1988)). The band at the bottom of FIG. 1 is IgG.Molecular weight markers in kDa are indicated.

rtrk 3T3 cells were generated by CaPO₄ mediated transfection of a rattrk CDNA into NIH-3T3 cells. Rat trk cDNAs were obtained from anembryonic rat DRG CDNA library kindly provided by M. C. Fishman. Thelongest trk cDNA obtained (2.4 kbp) was missing approximately 150 bp ofthe coding region as compared to available mouse and human trk sequence.The missing bases plus minimal (˜50 bp) 5' flanking non-coding sequenceswere replaced from mouse first coding exon sequences and thereconstructed gene was placed downstream of an MSV-LTR. PC12 cells orrtrk 3T3 cells (2×10⁷) were labeled with ³² P orthophosphate (1 mCi/mlin 4 ml) for 4 hours at 37° C. Cells were treated with NGF for theindicated times, washed, lysed in buffer containing 1% NP40, and thelysates immunoprecipitated with trk antibody 43-4 (Kaplan et al., Cell61:125-133 (1990)) and electrophoresed on 7.5% SDS-PAGE gels aspreviously described (Kaplan et al., (1990)). For b, the phosphorylatedtrk bands were eluted from the gel and phosphoamino acid analysisperformed as described (B. M. Sefton et al., J. Cell 24:165-174 (1981)).p140trk protein from NGF treated PC12 cells was phosphorylated in vivo.For c, rtrk 3T3 cells were treated with 1 mM suramin in Dulbecco'sModified Eagle Medium (DMEM) for 2 hours or mock treated. Followingextensive washing of the cells with DMEN, NGF was added for the timeindicated cells were lysed and the lysates were immunoprecipitated withtrk antibody. Inmunoprecipitates were either subjected to immunoblotanalysis with the phosphotyrosine (Ptyr) monoclonal antibody 4G10 (Lanes1-3), or were analyzed in kinase assays. (Morrison, et al. Cell58:649-657 (1989) and Kaplan et al. (1990)) Similar amounts of & proteinwere present in each lane.

FIG. 2 shows the time course, growth factor specificity, and doseresponse of trk tyrosine phosphorylation in PC12 cells.

a, Time course of trk tyrosine phosphorylation. Cells (2×10⁷) weretreated with 50 ng/ml NGF at 37° C.

b, Effects of growth and differentiation factors on trk tyrosinephosphorylation. Cells were treated with 100 ng/ml NGF, 100 ng/ml basicfibroblast growth factor (FGF) (Boehringer Mannheim Biochemicals), 100ng/ml epidermal growth factor (EGF) (Upstate Biotechnology, Inc.), 100nM insulin (Signman), or 1 μg/ml phorbal 12-myristate 12-acetate (PMA)(Sigma) for 5 minutes at 37° C.

c, Dose response of trk tyrosine phosphorylation. Cells were treated for30 minutes at 37° C. with increasing concentrations of NGF. Shown areWestern blot analysis with Ptyr antibodies of trk immunoprecipitatesprepared with trk antibody 43-4.

FIG. 3 represents the trk expression in day 17 mouse embryo DRGs. a,Brightfield and b, darkfield optics of a sagittal section through thethoracic region of an E17 embryo. In sites protocols and probes havebeen described in detail elsewhere (Martin-Zanca et al. (1990)).

FIG. 4 shows NGF-dependent tyrosine phosphorylation of p140trk in thehuman neuroblastoma cell lines LA-N-5, SY5Y, and dorsal root gangliafrom mouse embryos. a, p140trk was immunoprecipitated from untreated (-)or NGF treated (+) LA-N-5 cells (K. H. Sonnenfeld and D. N. Ishii, J.Neurosci. Res 8:375-391 (1982)), SY5Y cells (Sonnenfeld and Ishii,(1982)), NR18 cells (M. A. Bothwell et al., Cell 21:857-866 (1980)), orPC12 cells. Immunoprecipitates were probed with P-tyr antibodies. Thedifferences in trk protein mobilities are due to differences inglycosylation. b, tyrosine phosphorylation of p140trk in DRGs from 13.5day or 14.5 day embryonic mice. DRGs were maintained in 100 ng/ml NGFfor ≧10 minutes prior to lysis and immunoprecipitation with trkantibodies. Trk immunoprecipitates were probed with P-tyr antibody.Tyrosine phosphorylated p140trk from NGF-treated (+) PC12 cells oruntreated (-) PC12 cells is shown for comparison. Samples werenormalized for cell protein. Molecular weight markers in kDa areindicated.

Cell lines were treated with 100 ng/ml NGF for 5 minutes and p140trkimmunoprecipitated as in FIG. 1. DRGs were prepared by dissection for13.5 or 14.5 day mouse embryos. 100 DRGs were treated with NGF, washed,and subjected to Dounce homogenization in 1% NP40 lysis buffer. Lysateswere immunoprecipitated with trk antibody and the trk proteins wereanalyzed by antiphosphotyrosine immunoblots.

FIG. 5 depicts the affinity crosslinking of NGF to p140trk on PC12 cellsand rtrk 3T3 cells. Trk receptors were labeled by cross-linking of ¹²⁵I-NGF to cultured cells using N-hydroxylsuccinimidyl-4-zidobenzoate(HSAB). The cell lines analyzed were PC12 cells (lanes 1-4), rtrk 3T3cells (lanes 5-9), NIH-3T3 cells (lanes 10-11) and A875 human myelomacells (lanes 13-15). Lysates from cells were immunoprecipitated withanti-NGF (lanes 1, 5, 9-14), p140w antibody 7-4 which is another trkantibody generated in bacteria against the p70w oncogene (7-4),(Martin-Zanca, et al., (1988)) (lanes 2 and 6), or p140trk antibody 43-4in the absence (lanes 2, 7 and 15) or presence (lanes 4 and 8) of 10μg/ml competing trk peptide. Crosslinking was performed in the presenceof excess unlabeled NGF (5 μm) in lanes 9, 11, and 14. The antibody 7-4immunoprecipitates 3-5 fold less p140trk than does antibody 43-4.Molecular weight markers in kDa are indicated.

¹²⁵ I-NGF was prepared by lactoperoxidase treatment to specificactivities of 2500-3500 cpm/fmole. Crosslinking of p140trk to ¹²⁵ I-NGFwas performed as previously described (B. L. Hempstead, Science243:373-375 (1989)). Cells (2×10⁶ /ml) were incubated with 0.5nM ¹²⁵I-NGF for 2 hours at 4° C. HSAB (50 μM) was added and the reactionexposed to long ultraviolet wavelight for 10 minutes. After washing in50 mM lysine in phosphate buffered saline, cells were lysed in buffercontaining 1% NP40 and the lysates immunoprecipitated and analyzed on7.5% SDS-PAGE as described (Kaplan et al., (1990)).

FIG. 6 demonstrates the equilibrium binding analysis of trk receptors incell membranes prepared from rtrk 3T3 cells. Binding of ¹²⁵ I-NGF wasanalyzed in crude membrane preparations by filter binding as described(Hempstead (1989)). Reactions were carried out in triplicate in thepresence or absence of excess unlabeled NGF with 10 μg of membraneprotein for 1 hour at 30° C. and filtered under vacuum through MilliporeHVPL filters. Over 80% of specific binding was detected aftersubtracting values obtained in the presence of unlabeled NGF.

FIG. 6A is a saturation binding curve. FIG. 6B is data in (A) plottedaccording to Scatchard. Only binding values above 50% specific bindingwere used. The LIGAND program was used to determine Kd.

FIG. 7 depicts Northern transfer analysis of trk and trkb transcripts inNGF treated (+) or untreated (-) PC12 cells. RNA preparation andNorthern transfer analysis was performed as described previously (D.Martin-Zanca et al., Genes Dev. 4:683-694 (1990)). Cells were treatedwith 50 ng/ml NGF(+) (Boehringer Mannheim Biochemicals) and wereharvested 48 hours later after differentiation had occurred. 20 μg oftotal RNA was loaded per lane, and the filter was hybridized with a trk(Martin-Zanca et al., (1990)) or trkb (Klein et al., Development4:845-850, (1990)) specific probe.

FIG. 8. Treatment with K-252b stimulates NT-3 induced neurite outgrowthand trk tyrosine phosphorylation in PC12 cells while inhibiting NGFinduced effects. PC12 cells were incubated for 48 hours with K-252b (50nN) and NT-3 (50 ng/ml) or NGF (50 ng/ml).

A. The percentage of cells with neurites after 48 hours incubation isshown. Neurites were scored if they were a length of one cell body ormore.

B. PC12 cells were subsequently lysed and immunoprecipitated withanti-trk serum. Trk proteins were subjected to SDS-PAGE and analyzed byimmunoblotting with anti-phosphotyrosine antibodies. Lane 1,NT-3+K-252b: lane 2, NT-3; lane 3, NGF+K-252b; lane 4 NGF.

C. PC12 cells were incubated for 1 hour in K-252b (50 nM or 10 μM) andwith NT-3 or NGF for 5 minutes prior to lysis. Other methods were thesame as in B. Lane 1, NT-3+10 μM K-252b; lane 2, NT-3+50 nM K-252b; Lane3, NT-3; lane 4, NGF+10 μM K-252b; lane 5, NGF+50 nM K-252b; lane 6,NGF; lane 7, untreated control.

FIG. 9. K-252b interacts directly with the trk and trkB protein. Humantrk and trkB were expressed in Sf9 insect cells transfected using abaculovirus system (R. Stephens and D. R. Kaplan, unpublished). The trkand trkB proteins were immunoprecipitated with trk antibody and theimmunoprecipitates were incubated with 20 μCi gamma-³² P!ATP, 10 mMMnCl₂, 20 mM Tris pH 7.4, for 5 minutes at 25° C. in the presence ofincreasing amounts of K-252b or DMSO alone. Phosphorylated proteins weresubjected to SDS-PAGE and the phosphorylated trk and trkB proteins werevisualized by autoradiography. The tyrosine kinase activity of the trkand trkB was activated in the absence of ligand, a common observationfor receptor tyrosine kinase produced in the baculovirus system. trk andtrkB proteins were 25% pure as assayed by SDS-PAGE. The positions of trkand trkB on the SDS-PAGE gel are shown.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to a complex comprising nerve growthfactor (NGF) and trk-proto-oncogene protein. The present inventionfurther relates to methods of utilizing the complex.

One embodiment of the present invention relates to a complex formed bythe interaction of NGF with trk-proto-oncogene protein that is free ofthe protein with which it is naturally associated. Thetrk-proto-oncogene product is a 140 kDa. glycoprotein tyrosine kinaseand a component of the high affinity NGF receptor.

The present invention relates to detection and quantitation methods thatcan be used in diagnostics and therapeutics in identifying NGF (ligand),trk-proto-oncogene protein receptor or the ligand-receptor complex.

Neurons of the central and peripheral nervous system are dependent onNGF for their continued survival. To date, NGF-dependent neurons thathave been identified are sensory neural crest-derived (trigeminal,superior, jugular and dorsal root ganglia neurons), sympathetic neuronsand cholinergic neurons of the basal, media septal and diagonal septalband nuclei of the brain. This last neuronal type are found to bedegenerative in Alzheimer's and Huntington's diseases.

The knowledge and understanding of NGF-mediated responses as occurringvia a complex with the trk tyrosine kinase has broad implications forthe study of nerve survival, regeneration and accurate diagnosis andpotential therapy for neurodegenerative diseases that affectNGF-dependent neurons.

Since NGF-dependent neurons respond via the NGF-trk proto-oncogenetyrosine kinase complex, the methods described herein provide a meansfor identifying other neuronal types other than those described abovewhich will lead to the identification of other neuronal disorders. Inthis regard, applicants have recently identified trk expression (andtherefore NGF-responsive neurons) in the trigeminal mesencephalicnucleus. These neurons mediate many important sensory functionsthroughout the brain and can be affected in as yet unidentified neuronaldisorders.

The methods of the present invention will also aid in the understandingof the role of the interaction between NGF and its receptor, thetrk-proto-oncogene product as a transducer of NGF signals. Considerableexpertise and information is available from the past study of tyrosinekinases in other biological systems (i.e., oncogenesis and cell growth)that indicate existing biochemical cascades within the cell that are thesignal transducing pathways to the nucleus. Thus NGF binding to trkinitiates a signal cascade inside the cell that is amendable toidentification, study, and perhaps ultimately, to manipulation,utilizing skills and methodologies that are already in existent.

The present invention further relates to a method of detecting andquantitating trk-proto-oncogene receptor in a biological sample usinglabeled. NGF as a probe. Suitable labels include, for example,radiolabels such as ¹²⁵ I, and fluorescein.

Using standard methodologies well known in the art and described herein,a biological sample can be extracted with a non-ionic detergent andincubated with labeled NGF in the presence or absence of unlabeled NGF.The resulting complex can be separated from the uncomplexed (or unbound)labeled material, for example, by immunoprecipitating the complex with aspecific polyclonal antibody, for example, the 43-4 or 7-4 rabbitanti-trk antisera and in parallel, monoclonal phosphotyrosine antibody,such as Ptyr 4G10, for example, that recognizes the trk-proto-oncogenereceptor protein or the NGF-trk proto oncogene receptor complex. Theoverall signal resulting from the presence of label associated with theresulting complex is compared with the signal from a mock sample. Themock sample is prepared using purified trk-proto-oncogene receptorprotein in a known quantity treated the same way as the biologicalsample.

Alternatively, the complex can be separated from uncomplexed material byprecipitating with polyethylene glycol. In both methodologies, theamount of label that is immunoprecipitated or precipitated is directlyrelated to the amount of complex in the biological sample.

The present invention also relates to a method for detecting andquantitating NGF in a biological sample using labeled trk-proto-oncogenereceptor as a probe. The method is carried out as a reciprocal bindingassay following the methodology described above except substituting asantibody, one that specifically recognizes NGF or theNGF-trk-proto-oncogene receptor complex. Antibodies against NGF are wellknown in the art.

The present invention also relates to further methods of detecting andquantitating NGF-trk-proto-oncogene protein receptor complexes in asample. In one aspect, complexes are detected and quantitated usingantibodies directed against NGF, proto-oncogene receptor protein or theNGF-receptor complex. Antibodies can be either polyclonal or monoclonal;examples of both are described above and below in the Example Section. Asample can be extracted with non-ionic detergent and incubated withlabeled NGF or trk-proto-oncogene receptor protein. After incubation,the sample is covalently cross-linked with a lipophilic photoaffinitycross-linking agent for example, HSAB. Chemical crosslinking agents,such as disuccinimidil suberate (DSS) can also be used in thisprocedure. The sample is immunoprecipitated with specific antibody orprecipitated with polyethylene glycol. Quantitation requireschromatographic separation by, for example, gel electrophoresis,followed by autoradiography.

The present invention also relates to diagnostic methodology using themethods described above. The disorders which are diagnosed by themethods of the present invention include, for example, neurodegenerativediseases that affect NGF-dependent neurons such as Alzheimer's andHuntington's diseases. The present diagnostic methods can also be usedto measure neuronal disorders in tissue derived from neuronal cell typesdescribed previously, which will probably lead to diagnostics of as yetunidentified neuronal disorders.

The present invention further relates to methods of detecting other trkrelated receptor and NGF related neurotrophic factor complexes usingsimilar methods as those utilized above for detecting the trk/NGFcomplex. The trk gene is a member of a structurally related gene familyof which at present at least three members have been identified (trk,trkb, and trkc). Likewise a growing number of neurotrophic factors areemerging on the basis of similar structure and function to NGF such asBDNF and NT-3 for example. It is very likely that methods used toidentify the trk/NGF complex will lead to parallel discoveries with theadditional trk and NGF-related genes. The strategies used to identify,characterize and study these trk-related/NGF-related complexes (ie.:trkb/BDNF) will be based on the discovery herein described. Anyimplications at the practical or therapeutic levels will apply to theseneurotrophic factors. The knowledge of trk-related/NGF-relatedcomplexes, for example, Trkb/BDNF, will provide insight into thesurvival capacities of a different subset of nerve cells to thosedependant on NGF. Assays and strategies similar to those previouslydescribed conceived or devised for detecting the trk/NGF complex wouldapply to the detection of the related complex for example, use ofphosphotyrosine and trkb antibodies for immunoprecipitatingtrk-related/NGF-related complexes.

In an additional embodiment the present invention relates to a methodfor measuring nerve regeneration in trk proteins by measuring trkphosphorylation comprising the steps of adding a compound to said trkproteins in the absence or presence of a neurotrophic factor;precipitating the trk proteins with antibodies that recognize trkproteins; subjecting the precipitated trk proteins to electrophoresis;transferring the electrophoresed trk proteins onto a membrane; andmeasuring the amount of phosphotyrosine on the membrane.

In a further embodiment, the present invention relates to a method formeasuring nerve regeneration in trk proteins by measuring trkphosphorylation comprising the steps of combining a compound with trkprotein, a neurotrophic factor and ³² P labeled ATP to form a reactionmixture; incubating the reaction mixture; removing excess ³² P labeledATP from the mixture; subjecting the mixture to gel electrophoresis; andmeasuring the amount of phosphotyrosine on the gel.

The present invention further relates to therapeutic methodologies andthe development of detection kits or pharmacological agents that enhanceNGF-mediated nerve regeneration or survival. This will depend on the useof trk antibodies and phosphotyrosine antibodies to assay for thequality of the procedure. Most obvious in the area of potentialtherapeutic value is the development of drugs that either enhance orinhibit tyrosine phosphorylation. Since trk mediates signalling viaphosphorylation on tyrosine of messenger molecules, its signalling couldbe altered as required in cells. These studies would initially bedeveloped and assessed in tissues or cell culture systems prior to anypotential application. Drugs would be added to trk-expressing tissueculture cells together with or in the absence of NGF and the state oftrk activation, as measured by tyrosine phosphorylation, could beassessed. Progress in developing these drugs would be most effectivelymonitored with antibodies that recognize trk and/or phosphorylatedtyrosines. Thus development of any useful therapies in this area willdepend on the ability to identify the activation state of trk and/or anyof its downstream substrates. Next, animal models (rat or mouse) will beused in which specific nerve connections are disrupted, the promisingpharmaceuticals administered, and finally analysis of the sacrificedanimals is performed to assess the regeneration of nerves using trk/NGFor trk-related NGF-related antibody assays as described.

The present invention also relates to other therapeutic methods fordesigning pharmaceuticals that enhance the stimulation of degenerativenerves in diseases such as Alzheimer's and Huntington's.

Trk and low affinity NGF receptor 75kNGF-R are required together forhigh affinity response to NGF. Methods could be devised that wouldenhance detection of NGF using the high affinity complex. Knowledge ofthe existence for a trk/NGF complex could lead to the development ofmodified NGF molecules that hyperstimulate & activation. These NGFderivatives might be of importance in the stimulation of degeneratingnerves stemming from diseases, for example, Alzheimer's andHuntington's, or from injury.

Many substrates of tyrosine kinases have been identified. Identificationof trk-specific substrates could lead to the discovery of anintermediate molecule in the NGF pathway that can be manipulatedpharmacologically to enhance or inhibit NGF-mediated signals.

The finding of parallel effects of K-252b on neurite outgrowth and trktyrosine auto-phosphorylation in PC12 cells in the presence of NT-3 orNGT, respectively, suggest that the selective inhibitory and stimulatoryactions of K-252b on neurotrophin effects involve interaction withtrk-type receptor mechanisms. It remains to be determined, however,whether such interaction is by direct effect on protein kinase activityof the trk protein. A possible explanation for the observation ofincreased NT-3 effects is that K-252b modifies trk in a manner that thisreceptor will interact with NT-3, but not NGF, more efficiently. WhileK-252a does not interfere with binding of ¹²⁵ I-NGF to PC12 cells(Koizumi et al. (1988)), it seems possible that the related K-252b mightaffect binding of selected neurotrophins to specific active sites. Suchan effect could also involve other proteins which are believed to bepart of neurotrophin receptors, like the p75-NGFR low affinity NGFreceptor protein (A. Rodriguez-Tebar et al, Barde, Neuron 4, 487-492(1990)); (M. Bothwell, cell 65:915-918 (1991)).

K-252b inhibits protein kinase C, as well as cAMP- and cGMP-dependentprotein kinases with K values in the 10-100 nM range as shown by invitro assay systems (Kase et al. (1987)). Given this rather broadspectrum of inhibitory actions, the selective inhibition of neurotrophineffects is surprising. The two related molecules K-252b and K-252ainhibit similar protein kinases with only slightly different substratespecificities in cell-free assay systems (Kase et al. (1987)), whereasthere are pronounced differences between their actions on intact cells(Knusel and Hefti (1991)). In contrast to K-252a, K-252b is ahydrophilic molecule with a free carboxylic acid residue and, therefore,does not freely pass the cell membrane (K. Nagashima, S. Nakanishi, Y.Matsuda, Febs Letts. in press (1991)). Because of this property, theintracellular concentration and distribution of K-252b in differentcompartments of living cells will probably be very different from thatof K-252a. The selectivity of the effects on neurotrophin actions couldbe explained if only very limited amounts of K-252b enter the cells andif K-252b is more potent in modifying trk protein kinase activity thanother protein kinases, including other growth factor receptor kinases.Alternatively, K-252b, in intact cells, might interact with anecto-protein kinase (Y. H. Ehrlich et al., Nature 320:67-70 (1986)) or,specifically, with extracellular or transmembranal domains of trkproteins, without access to intracellular protein kinases. Thispossibility is supported by recent findings showing that K-252b inhibitsthe protein kinase activity of the platelet-derived growth factorreceptor in cell-free preparations but not in intact cells (D. R.Kaplan, unpublished observations). Different cellular distribution mightalso explain the absence of neurotrophic-like effects of K-252b as theyare observed with K-252a in which DRG cells (Borasio (1990)) or in ratdopaminergic neurons (Knusel and Hefti (1991)), or with staurosporine inPC12 cells (S. Hashimoto and A. Hagino, J. Neurochem. 53:1675-1685(1989)); (A. S. Tischler et al., J. Neurochem. 55:1159-11 6 (1990). Thediscovery of NT-3 potentiation by K-252b invites the explanation thatneurotrophic-like effects of K-252a and staurosporine could involveenhancement of the action of an endogenous neurotrophin which might bepresent in the respective cultures at low concentration. If thisexplanation is true, this neurotrophin would be different from BT-3since no similar trophic effects have been found with K-252b in absenceof exogenously provided trophic protein (Knusel and Hefti (1991)). Thedose requirements for NT-3 potentiation on basal forebrain cholinergicneurons by K-252a and K-252b seem to be identical, whereas the lattercompound is approximately threefold less potent in inhibiting NGFeffects on the same neurons (Knusel and Hefti (1991)). This observationsuggests that the structural requirements for NT-3 stimulation and forNGF inhibition are different, evidence being added by the fact thatstaurosporine, which inhibits NGF actions on the same neurons with highpotency (Knusel and Hefti (1991)), does not enhance the NT-3 action.

Applicants' findings suggest K-252b as a valuable tool to study themechanisms of action of neurotrophins and to demonstrate biologicalactions of these proteins in vivo and in vitro. Compared to the relatedcompounds, K-252a and staurosporine, which show complex patterns ofactivity and are cytotoxic (Knusel and Hefti (1991)), K-252b is anon-toxic and highly selective modifier of neurotrophin actions invitro. Most importantly, comparison of the results with threestructurally related inhibitors suggest that the requirements for NGFinhibition and for NT-3 potentiation are different from each other.These findings raise the possibility of development of compounds of evenhigher selectivity, able to inhibit or potentiate action of individualneurotrophins. They identify K-252b as leading compound for furtherstructural modifications and the development of other selectivemolecules. Such molecules would be valuable tools to investigatebiological functions of neurotrophins. Furthermore, it is likely that,in the future, highly selective drugs with agonistic or antagonisticactions on neurotrophin mechanisms will become therapeutically useful inthe treatment of neurodegenerative diseases and nervous system injuryand, even pathological cell proliferation.

EXAMPLES Example 1

Tyrosine phosphorylation of p140^(PROTOTRK) in response to NGF

The stimulation of p140^(prototrk) tyrosine phosphorylation in responseto NGF addition to PC12 cells is rapid, specific and occurs in thepresence of physiological amounts of NGF.

This previous study utilized immunoblotting analysis withphosphotyrosine (P-tyr) antibodies to detect tyrosine phosphorylation ofp140^(prototrk). To determine if enhancement of serine or threoninephosphorylation of p140^(prototrk) are induced by NGF, and to comparethe relative amounts of tyrosine, serine, and threonine phosphorylation,PC12 cells were labeled with ³² P-orthophosphate prior to NGF treatmentand immunoprecipitation with antibodies to p140^(prototrk).p140^(prototrk) was phosphorylated predominately on serine residues inimmunoprecipitates from untreated cells and cells treated with 50 ng/mlNGF for 5 minutes. The presence of NGF, however, stimulated the tyrosinephosphorylation of p140^(prototrk) 20-fold, although this representedless than 5% of the newly incorporated phosphate residues. In contrast,p140^(prototrk) was labeled predominantly on tyrosine in immune complexkinase assays from NGF-treated PC12 cells or in ³² P-labeled NIH-3T3cells transfected with the rat trk gene (rtrk-3T3) (FIG. 1a). Thetyrosine phosphorylation of p140^(prototrk) expressed in NIH-3T3 cellswas constitutive, apparently due to autocrine stimulation by NGFproduced by these cells. Treatment of rtrk-3T3 cells with suramin, apolyanionic compound which inhibits and reverses the binding of somegrowth factors to their receptors (M. Hosang et al., J. Cell. Biochem.29:265-273 (1985)), markedly reduced the tyrosine phosphorylation ofp140^(prototrk) in vivo and in immune complex kinase assays (FIG. 1b).When NGF was added to the suramin treated cells for 10 minutes, tyrosinephosphorylation of p140^(prototrk) observed in vivo and in vitro wasstimulated at least 10-fold (FIG. 1b).

Trk tyrosine phosphorylation occurred within one minute of NGF treatmentcells, reached maximum levels after five minutes, and declinedthereafter (FIG. 2a). Residual phosphorylation was detected after twodays of treatment with NGF when the cell population was fullydifferentiated. Trk tyrosine phosphorylation was also specific to NGF.Other peptide growth factors that elicit tyrosine phosphorylation inPC12 cells were tested in this assay (V. Hamburger and R.Levi-Montalcini, J. Exp. Zool. 111:457-502 (1949)); (I. B. Black et al.,Growth Factors and Development, Current Topics in Developmental Biology,Vol. 24: (ed. Nilsen-Hamilton, M.) 161-192 1990)). EGF, basic FGF,insulin, and the phorbol ester PMA failed to induce trk; however, trkwas induced in cells treated with NGF or the combination of basic FGFand NGF (FIG. 2b). It has been previously shown that these agentsproduce similar patterns of early responses in PC12 cells, includingtranscriptional activation of c-fos and cmyc (M. E. Greenberg et al., J.Biol. Chem. 260:14101-14110 (1985)). However, only NGF and basic FGFstimulate neurite outgrowth.

To determine the minimal concentration of NGF capable of eliciting trktyrosine phosphorylation, a dose response experiment was performed.Tyrosine phosphorylation was half maximal at 0.1 ng/ml NGF (50 pM) (FIG.2c) indicating the trk phosphorylation occurs at physiologicallyrelevant concentrations of NGF (S. Cohen, Proc. Natl. Acad. Sci. USA46:302-311 (1960)).

Example 2

Expression of trk gene in embryonic sensory neural crest-derived neurons

The trk gene is expressed in embryonic sensory neural crest-derivedneurons including dorsal root ganglia (DRG) (FIG. 3 and Martin-Zanca etal. 1990). This expression is confined to neurons (note that the darklystaining glial cells are devoid of silver grains) and maintained in theadult. To determine whether the trk protein in embryonic neurons wasresponsive to NGF, DRG from E13.5 and E14.5 mouse embryos wereexplanted, maintained in 50 ng/ml NGF on ice for 10 minutes, lysed, andsubjected to trk antibody precipitation and anti-ptyr immunoblottinganalysis. As shown in FIG. 3A, phosphorylation of the p145^(prototrk)was detectable in 14.5 day DRG but not in two independent preparationsof 13.5 day DRG. Tyrosine phosphorylated trk protein was not detectablein the absence of exogenously administered NGF.

Dissection of DRG provides primarily the cell bodies and eliminates theaxons, therefore the significance of these data with regard to timingand degree of p145^(prototrk) activation should be interpreted withcaution. The results in 14.5 DRG, however, demonstrate that freshlydissected embryonic DRG neurons contain trk protein which isphosphorylated in response to NGF.

Example 3

NGF stimulates p140^(prototrk) tyrosine phosphorylation in severaltrk-expressing cell types

To determine whether phosphorylation of p140^(prototrk) in response toNGF was unique to rat PC12 cells or occurred in other NGF responsivecell lines, the state of the trk protein in additional neuroblastomacell lines from different species was assayed. It was observed thatp140^(prototrk) tyrosine phosphorylation was also enhanced by NGF in thehuman neuroblastoma cell line LA-N-5 and in the murine cell line SY5Y(FIG. 4b). LA-N-5 and SY5Y cells express 4-fold less trk mRNA than PC12cells, accounting for the lower amounts of tyrosine phosphorylated trkobserved in these cell lines compared to PC12 cells.

Derivatives of the PC12 cell line have been generated by autagenesisthat have lost high affinity response to NGF (Bothwell et al. 1981). Onesuch line, NR18, lacks 75kNGF-R. Introduction of 75kNGF-R into thesecells resulted in the reconstitution of biphasic Scatchard profile andat least partial function reconstitution (Hempstead et al. J. Biol.Chem. 265:9595-9598 (1990)). NR18 cells express the trk proto-oncogeneat greatly reduced levels (see Hempstead et al. 1991).

Applicants next analyzed the phosphorylation state of the trk receptoron the NR18 cell line that has greatly reduced responsiveness to NGF(Bothwell et al., Cell 21:857-866 (1980)). Consistent with RNAexpression data (see Hempstead et al. 1991) no phosphorylation ofp140^(prototrk) in response to NGF was observed in these cells (FIG.4b). Thus, in NR18 cells, the tyrosine phosphorylation ofp140^(prototrk) correlates with the reduced ability of NGF to elicit abiological response.

Example 4

Trk receptor directly binds to NGF

The above results, demonstrating the rapid phosphorylation ofp140^(prototrk) in several trk-expressing cell lines treated with NGF,suggested that the trk receptor might directly bind NGF. To determine ifNGF was capable of binding to p140^(prototrk), several cell lines wereanalyzed for the ability of trk-specific antisera to precipitatereceptor-ligand complexes in affinity crosslinking experiments (FIG. 5).The cell lines assayed were rat PC12, human LA-N-5, mouse SY5Y, mouseNIH-3T3, mouse rtrk-3T3, and human AB75 cells. NGF induces the tyrosinephosphorylation of p140^(prototrk) in PC12, LA-N-5, SY5Y, and rtrk-3T3,but not in AB75 melanoma or NIH-3T3 cells which express no detectabletrk messenger RNA. ¹²⁵ I-labeled NGF was crosslinked to cells using thelipophilic photoaffinity agent HSAB. Previous studies with thiscrosslinking agent have shown that in PC12 cells and sympatheticneurons, two NGF containing species of 100 kDa and 150-160 kDa areobserved (J. Massacue et al., J. Biol. Chem. 256:9419-9424 (1981);Hempstead, et al. 1990; S. O. Meakin and E. M. Shooter, Neuron 6:153-163(1991)). The 100 kDa species represents ¹²⁵ I-NGF bound to 75kNGF-R (M.Hosang and E. M. Shooter, J. Biol. Chem. 260:655-662 (1985)). Followingcrosslinking, the cells were washed to remove unbound ¹²⁵ I-NGF, lysedin detergent, and the lysates incubated with antibodies (FIG. 4). It wasobserved that the 160 kDa species in anti-NGF or anti-p140^(prototrk)immunoprecipitates from PC12 and rtrk-3T3 cells, and not in A875 orNIH-3T3 cells. The immunoprecipitation of the 160 kDa species wasblocked by addition of a trk-derived peptide used to generate theantibody, and was not seen if excess unlabeled NGF was added to the ¹²⁵I-NGF treated cells prior to crosslinking. A 160 kDa crosslinked productwas also observed in LA-N-5 and SY5Y cells. The crosslinked 100 kDaspecies were present in PC12 and A875 cells and not in the 3T3 celllines, reflecting the absence of expression of the 75kNGF-R in NIH-3T3cells. The above experiments establish that NGF binds to p140^(prototrk)and that this binding is seen only in cell lines which showp140^(prototrk) tyrosine phosphorylation in response to NGF.

Of equal importance to the demonstration of binding, it is essential todetermine whether the affinity of binding reflects physiologicallyrelevant conditions. Scatchard plot analysis was carried out todetermine the affinity of NGF for p140^(prototrk) expressed in NIH-3T3cells. Crude membranes were prepared from cells and assayed by bindingto ¹²⁵ I-NGF. Membranes obtained from rtrk-3T3 cells displayed a linearScatchard plot with a Kd of approximately 10⁻⁹ M (FIG. 6). By thisanalysis, the number of receptors was approximately200,000-500,000/cell.

Example 5

Expression of trk or trk-related messenger RNA in several cell types

The trk gene is a member of a gene family of TK receptors that includesthe related gene trkb. To determine if trk is transcribed in PC12 cells,the expression of trk transcripts was assayed by Northern transferanalysis with a full-length trk cDNA probe (R. Klein et al., Development4:845-850 (1990)). PC12 cells contained trk transcripts (FIG. 7). Thelevel of trk transcripts was not affected by the addition of NGF. Todetermine whether additional trk-related genes were transcribed in PC12cells, mRNA was hybridized at low stringency with the highly conservedtrk TK domain. Trk transcripts have been found in LA-N-5 cells, Sy5ycells and DRG from 13.5 day or 14.5 day embryonic mice. Trkb expressingcell lines, as determined by mRNA analysis will help determine the nextsteps in interactions with trkb ligand (BDNF)

Example 6

Neurite outgrowth and trk phosphorylation in PC12 cells

Experiments on PC12 cells confirmed observations made on primary neuroncultures. PC12 cells were grown as described earlier (Kaplan et al.(1990), (1991a), (1991b)). To assess effects on neurite outgrowth, cellswere incubated for 48 hours with K-252b (50 nM) and NT-3 or NGF (50ng/ml). Neurites were scored if they were a length of one cell body ormore. To measure the status of trk tyrosine phosphorylation, PC12 cellswere lysed and immunoprecipitated with anti-trk serum. Trk proteins weresubjected to 7.5% SDS-PAGE and analyzed by immunoblotting withantiphosphotyrosine antibodies as described in detail elsewhere (Kaplanet al. (1990), (1991a), (1991b)). Tyrosine phosphorylation of cellularproteins was assayed by probing Western blots of lysates of PC12 cellsincubated with K-252b and NT-3 or NGF with anti-phosphotyrosineantibodies. Phospholipase C gamma 1 was identified in these blots asdescribed elsewhere (M. L. Vetter et al., Proc. Natl. Acad. Sci. USA88:5650-5654 (1991)).

PC12 cells incubated for 48 hours in the presence of NT-3 (50 ng/ml) andK-252b (50 nM) showed significantly more neurite outgrowth activity thancells incubated with NT-3 alone (FIG. 8A). At the concentration ofK-252b used in this experiment, NGF-induced neurite outgrowth wasreduced approximately three-fold. This result is similar to a previousobservation with K-252a. Higher concentration also of this inhibitor isrequired to block the NGF action in cholinergic neurons in primarycultures than to suppress NGF effects in PC12 cells (Koizumi et al.(1988)); Smith et al. (1989)); Knusel and Hefti (1991)).

Example 7

K-252a inhibits the tyrosine phosphorylation of trk in NGF-treated PC12cells

Experiments were conducted in which PC12 cells were treated with 0, 200or 500 nM of K252a prior to treatment of cells for 5 minutes with NGF.Trk proteins were immunoprecipitated with trk antibody, andelectrophoresed on polyacrylamide gels. Proteins were transferred tonitrocellulose filters and the filters were probed withanti-phosphotyrosine to observe trk tyrosine phosphorylation.

Example 8

Effects of K-252b on tyrosine phosphorylation of trk

The fact that K-252b inhibits various protein kinases in cell freesystems (Kase et al. (1987)); (Koizumi et al. (1988)); (Matsuda et al.(1989)) and the recent discovery that trk proto-oncogenes are involvedin the formation of high affinity neurotrophin receptors (Hempstead etal. (1990)); (Kaplan et al. (1990), (1991a), (1991b)); (Squinto et al.(1990), (1991)); (Soppet et al., (1991)); (Cordon-Cardo et al., (1991))suggested that K-252b exerts its neurotrophin inhibitory and stimulatoryactions by directly interfering with protein kinases of the trk proteinfamily. NGF has been shown to stimulate the tyrosine phosphorylation ofp140trk within minutes of addition to PC12 cells (Kaplan et al.(1991b)). NT-3 induces only low levels of trk tyrosine phosphorylationand neurite outgrowth in these cells (FIG. 8A) (Berkemeier et al.,(1991)). PC12 cells which were exposed to NT-3 or NGF for 48 hours inthe presence or absence of K-252b (FIG. 8A) were also analyzed for 140KD trk tyrosine phosphorylation (FIG. 8B). NGF (50 ng/ml) stronglystimulated trk tyrosine phosphorylation. No phosphorylation wasdetectable if the cells were treated with NT-3 alone (FIG. 8B).Similarly, cells treated with K-252b alone were identical to untreatedcontrols (data not shown). However, a clear increase in trk tyrosinephosphorylation was seen in cells grown in simultaneous presence of 50ng/ml NT-3 and 50 nM K-252b (FIG. 8B, lane 1). Similar enhancement oftrk tyrosine phosphorylation was observed when cells were acutelytreated for 1 hour with -252b followed, during the last 5 minutes ofK-252b treatment, with NT-3 (FIG. 8C). Again, stimulation of tyrosinephosphorylation by NT-3 alone was minimal but was greatly enhanced bythe simultaneous presence of K-252b. In this assay, NGF mediatedtyrosine phosphorylation was inhibited two-fold or five-fold by 50 nM or10 μM K-252b, respectively.

Tyrosine phosphorylation of other cellular proteins was examined in PC12cells treated with NT-3 or NGF in presence of K-252b. The tyrosinephosphorylation of phospholipase C gamma-1, a direct target of the trktyrosine kinase (Vetter et al., (1991)), and several other cellularproteins, was inhibited by 10 μM K-252b in NGF treated cells. Incontrast, the NT-3 mediated tyrosine phosphorylation of these proteinsin PC12 cells was enhanced by 50 nM of K-252b (data not shown). Thetyrosine phosphorylation of EGF receptors in PC12 cells treated with 100ng/ml EGF for 5 minutes was not inhibited by 10 μM K-252b (data nowshown).

Example 9

K-252b interacts directly with the trk and trkB protein

Human trk and trkB were expressed in Sf9 insect cells transfected usinga baculovirus system (R. Stephens and D. R. Kaplan, unpublished). Thetrk and trkB proteins were immunoprecipitated with trk antibody and theimmunoprecipitates were incubated with 20 μCi gamma-³² P!ATP, 10 mMMnCl₂, 20 mN Tris pH 7.4, for 5 minutes at 25° C. in the presence ofincreasing amounts of K-252b or DMSO alone. Phosphorylated proteins weresubjected to SDS-PAGE and the phosphorylated trk and trkB proteins werevisualized by autoradiography. The tyrosine kinase activity of the trkand trkB was activated in the absence of ligand, a common observationfor receptor tyrosine kinase produced in the baculovirus system. trk andtrkB proteins were 25% pure as assayed by SDS-PAGE. The positions of trkand trkB on the SDS-PAGE gel are shown in FIG. 10.

Applicants' findings demonstrate that K-252b selectively modifiesactions of the neurotrophin growth factor family. At nM concentrations,K-252b selectively potentiates the actions on NT-3. At μMconcentrations, the compound inhibits the actions of all threeneurotrophins tested, without interfering with the effects ofnon-neurotrophin growth factors.

While the foregoing invention has been described in some detail forpurposes of clarity and understanding, it will be appreciated by oneskilled in the art from a reading of this disclosure that variouschanges in form and detail can be made without departing from the truescope of the invention.

What is claimed is:
 1. A method of increasing neurite outgrowth,comprising the step of contacting cells of neural origin, said cellsexpressing trk-proto-oncogene receptor protein, with a mixture of NT-3and K-252b, such that neurite elongation is effected among said cells.2. A composition for increasing neurite outgrowth comprising a mixtureof NT-3 and K-252b.